Translation is a highly conserved process that is essential and common to all forms of life, allowing an organisms genotype to be expressed as a phenotype. The majority of mRNAs contain an untranslated leader sequence, upstream (5') to the protein-encoding sequence, that contain "signals" for binding ribosomes and initiating translation. In all translation systems, mRNA binds first to the small ribosomal subunit, aided by initiation factors, followed by the large subunit. Leaderless mRNA, a specific sub-type of mRNAs found in all domains of life, initiate translation from a start codon at the 5' end of the mRNA and in the absence of signals typically found within the leader sequence. The mechanism by which leaderless mRNAs are found and bound by ribosomes is not understood. Using highly purified populations of ribosomes, ribosomal subunits and initiation factors, we explore here the involvement of a non-conventional pathway for translation initiation. Specific associations and interactions between leaderless mRNA and ribosomes or ribosomal subunits are probed by a variety of ribosome binding assays and site-specific photocrosslinking experiments. The described experiments have significant implications for novel translational regulation and ribosome function. A detailed analysis of mRNA:ribosome or mRNA:ribosomal subunit interactions might allow for the development of antimicrobial agents with highly specific and unique targets. The interactions of ribosomes with leaderless mRNAs are likely to be different from interactions with leadered mRNAs and provide an excellent opportunity to probe mRNA:ribosome interactions and reveal novel ribosome functions. The widespread occurrence of leaderless mRNAs suggests that the results of our studies in Escherichia coli and Streptomyces lividans will help us understand leaderless translation in other systems.